Applications of TBNK Antibody Kits & Newest Improvement on Current Standards

Abstract

Lymphocyte cell subsets are major research targets for a huge range of medical and drug discovery research, including the perennial search for better cancer and HIV treatments, as well as more recent Covid-19 therapeutic targets. Recently developed, the ReadiUse™ 6-Color Human TBNK Antibody Kit is designed to provide both the percentages and absolute counts of the lymphocyte subsets T-cells, B-cells, and NK-cells, using gated laser lines commonly present in flow cytometers. This research-use-only (RUO) kit is intended to differentiate the main lymphocyte subsets commonly present in whole blood and PBMCs.

Introduction

Tracking lymphocyte cell populations is a common requirement for monitoring multiple immune disorders in health facilities, as well as long-term medical research projects. Optimized for flow cytometry, the ReadiUse™ 6-Color Human TBNK Antibody Kit has been developed to eliminate multiple ‘pain points’ of prior TBNK kits, for simpler sample analysis and improved consistency. Additionally, it will do so in a no-wash format, using lyophilized materials for ease of usage and permitting long term RT storage instead of refrigeration.

By having known numbers of dried multicolor beads, and eliminating several steps of the typical protocols for similar kits, inconsistency is reduced as well as time at the bench and the expense of peripheral materials.

Lymphocyte Cell Subsets

The immune system is an incredibly complex network of biochemical pathways and reactive cascades, and immunology is a huge component of medical research. In very general terms, there are 4 types of immunological cells that are the most prominent, generally termed as lymphocytes. These 4 subsets are: T-cells—which are further split into helper or cytotoxic types, B-cells, and Natural Killer (NK) cells.

There are myriad structural and operational differences between these cells, but one of the most common laboratory methods of differentiating them is by detecting the specific surface and transmembrane Cluster of Differentiation (CD) antigens. Numerically assigned in the order they were discovered or first described, these proteins are referred to consistently via their CD nomenclature (i.e. how the CD antigens are named) which is a universally adopted designation system sanctioned by the World Health Organization (WHO). This system was designed for the classification of monoclonal antibodies (mAbs) directed against epitopes on the surface molecules of leukocytes. For difficult to characterize cell surface molecules, or ones only recognized by a single mAb, they are given the designation 'CDw' in which the ‘w’ stands for ‘workshop’ (e.g. CDw60 or CDw156). If the surface molecule is well characterized (recognized by 2 or more mAbs), then they are assigned a distinct value, such as CD4, CD8 or CD45.

CD nomenclature is also used to describe lymphocyte and leukocyte subtypes. The presence or absence of a specific antigen from the surface of the particular cell population is denoted with "+" or "-" respectively. A "+" symbol added to a CD number indicates the presence of that molecule on a cell or cell population, whereas a "-" indicates its absence. For example, a 'CD45⁺/CD3^-' cell is one that expresses CD45, but not CD3. Cell populations can also be defined as ‘hi’ or ‘low’ indicating an overall variability in CD expression, particularly when compared to other cells being studied. This can also be written as ‘bright’ or ‘dim’, in reference to the strength of fluorescent signal from the fluorophores used to detect them.

Since immune cells express CD antigens at various stages of development or cell activation, they serve as reliable biomarkers for differentiating leukocyte populations and subpopulations. This technique is known as immunophenotyping. By using targeted fluorescent-labeled antibodies, these cell types can be efficiently sorted via flow cytometry. CD antigen expression frequently varies based on the presence or progression of multiple pathologies, including leukemia, and so are often used as diagnostic or prognostic metrics.

The common lymphocyte subsets can be generally described as follows:

T-cells

CD4-expressing leukocytes, also known as helper T-cells, trigger the immune response by recognizing pathogens and secreting cytokines in order to signal to other immune cells, including CD8-expressing leukocytes, commonly referred to as cytotoxic T-cells. Helper T-cells are not directly responsible for the attack of the pathogens, only for identifying targets. Cytotoxic T-cells destroy the infected cells.

B-cells

Primarily produced in the bone marrow, CD19-expressing leukocytes, or B-cells, are an intrinsic part of the immune cascade. These cells produce antibodies against infections specifically targeted to the specific pathogen as identified by the helper T-cells.

NK-cells

Natural killer cells, or NK-cells, are part of the innate immune system, and act as defense not only against foreign pathogens, but also against anomalous native cells throughout the body, such as precancerous and infected cells. These effector cells are of great interest in new types of vaccines.

NK-cells present a variety of surface cell markers, notably CD16 and CD56 among others. There are also Killer Immunoglobulin-like receptors (KIR), as well as natural cytotoxicity receptors (NCR), which are prominent research targets in their own right.

In humans, NK cells are typically defined as CD3^-CD56⁺ cells. This definition will be explained in further detail in the next section. Two different NK-cell subtypes have been identified: CD3^-CD56^dimCD16⁺ and CD3^-CD56^brightCD16^-. As a reminder, this common nomenclature of ‘dim’ and ‘bright’ refers to the expression levels of these proteins on the cells, with ‘dim’ signal being detected from low expression, and ‘bright’ from high.

Other model species have differing NK-cell definitions. For example, in mice, NK-cells are defined as CD3^-NK1.1⁺ cells. Three subsets are characterized based on the differential expression of Integrin alpha M/CD11b and CD27, including CD11^dimCD27^bright NK-cells, CD11b^brightCD27^dim NK cells, and CD11b^brightCD27^bright NK-cells.

Research and understanding of these immune cells is ongoing, and contribute to therapies and preventative vaccine development for human health concerns worldwide.

Kit Components & General Usage

The ReadiUse™ 6-Color Human TBNK Antibody Kit is optimized for flow cytometry, and has only 2 components, both of which can be stored at room temperature (RT).

Self-contained tubes (sets of 5 or 25 depending on kit size) containing the TBNK dried reagent. This reagent is a cocktail of fluorescently-labeled monoclonal antibodies directed against human CD3, CD4, CD8, CD16, C19, CD45, and CD56. A known number of dried counting beads are included, permitting absolute lymphocyte subset cell concentration determination without a washing step. This not only eliminates a common source of experimental inconsistency, but also reduces hands-on time at the bench. A viability stain is also included.

Vials (1 or 5 depending on kit size) of 10x concentrated lyse/fix buffer. The concentrated lysing buffer is simple to dilute prior to use, and permits thorough mixing of the experimental sample.

All of the labeled antibodies are murine (mouse) host monoclonal, with human reactivity, and were purified through affinity chromatography. The specific clones were chosen for their consistency and prominence in research literature, for easy reproducibility and compatibility with known protocols.

Sample Protocol

Depending on instrumental setup and experimental goals, this suggested protocol should be optimized. However, a good starting point, and one that should suit basic usage of the kit is below.

Collect fresh samples if at all possible, ideally within 24 hours of analysis.

Prior to beginning the assay, prepare the 1X Lysis/Fix Buffer by adding 3.0 mL of deionized water to one bottle of the 10X Lyse/Fix Buffer (Labeled as component B in the kit). Mix and relabel the bottle as 1X Lysis/Fix Buffer. This mixed buffer can be stored for up to 1 month at RT away from light. Handle the concentrated or mixed buffer with suitable protection, as it contains formaldehyde.

1. Prepare blood cell samples: either fresh blood collected in an EDTA tube or isolated PBMCs at a concentration no greater than 106 cells/mL can be used.
   

   Note

   Refrigerated or excessively aged blood samples, or those from individuals taking immunosuppressive medications, are likely to give skewed results. All blood specimens are considered biohazards. Handle them as if they are capable of transmitting infection and dispose of them with proper precautions and in accordance with governmental regulations.

2. Remove the desired number of reagent tubes from the pouch and reseal the pouch.
3. Thoroughly mix the sample and dispense exactly 50 µL of sample into the designated reagent tube.
   

   Note

   The accuracy of the sample dispense will affect the accuracy of the absolute cell concentration determined.

4. Gently vortex each tube for 30 seconds to ensure complete solubilization of the dried reagent. This allows for all of the beads in the reagent to be mixed with the sample, ensuring improved accuracy of the absolute counts.
5. Incubate for 20 minutes at room temperature. Protect the tube from direct light.
6. Add 450 µL of 1X Lysis/Fix Buffer to each tube and vortex for 10 seconds. Return tubes to the dark for at least 15 minutes.
7. Vortex sample tube thoroughly at low speeds and load onto cytometer for analysis. The ReadiUse™ 6-Color Human TBNK Antibody Kit is designed to be used in a Lyse/No-wash format.
   

   Note

   If the sample is washed before analysis, the ability to determine absolute cell concentrations will be lost.

8. Flow Cytometer Acquisition: start and operate flow cytometer according to manufacturer’s instructions. Adjust the threshold to minimize debris and ensure populations of interest are included.

Analyze the data using the appropriate cytometer-specific software.

Relevant Cell Surface Antigens & Conjugated Labels

Figure 1. Hierarchical representation of the different lymphocyte subsets.

Because the CD antigens used to identify leukocytes are expressed at varying degrees and cell types, multiparameter flow cytometric analysis utilizing two or more combinations of fluorescently labeled CD antibodies permits clear resolution. This is the reasoning, aside from benchtime efficiency, that the ReadiUse™ 6-Color Human TBNK Antibody Kit, as well as preceding versions of similar kits, is designed with so many colors.

The kit includes labeled antibodies specific for CD3, CD4, CD8, CD16, C19, CD45, and CD56, which will be used to sort the cell types. These particular proteins are known to be expressed on particular leukocyte types, and their fluorescent labels were selected carefully based on expected signal strength and anticipated target frequency.

A more comprehensive data table of the CD antigens most frequently used for flow cytometry immunophenotyping in both human and mouse models is available here.

The infographic (figure 1) represents a simple logic cascade, and is how the flow cytometer sorts (and counts) the various lymphocyte cells, based on the fluorescent signals from each of the labeled anti-CD antibodies present in the kit’s reagent cocktail.

Before beginning the acquisition of any of the dot plots, it will be necessary to adjust the channel thresholds. In simplest terms, this is the separation of the forward from the side-scatter signal, gating appropriately to minimize cell debris. The beads in the kit have low FSC, and so the channel threshold should be adjusted to collect these events. If the debris box is too large, some beads may be lost. Methodology will vary between instruments, but is typically a relatively simple adjustment. Once the instrument has been gated correctly, this will permit accurate readings of initial ‘all events’.

The initial proprietary viability stain NucPO-1 indicates the first event, marking all dead cells present in the sample. This signal will be detected in the violet channel of the flow cytometer, to minimize overlap with other wavelengths. The second step is detection of the signal from CD45, a receptor-type protein which marks all lymphocyte types regardless of category. This requires a bright signal, and so the anti-CD45 antibody (clone 2D1) has been conjugated to PerCP-iFluor™ 680, for extremely bright and sustained fluorescence detected at 700 nm wavelength. By having such extreme difference in emission wavelengths between the viability and lymphocyte markers, crosstalk between them is minimized. CD45 is a glycoprotein also known as leukocyte common antigen (LCA), and is one of the larger proteins used in the kit, ranging from 180-240 kD.

Figure 2. Flow cytometry analysis of whole blood cells stained with iFluor® 488 anti-human CD3 antibody (Clone: SK7). The fluorescence signal was monitored using an Aurora flow cytometer in the iFluor® 488 specific B2-A channel.

The third step, which is also the first major gating, is marked by either the presence or absence of CD3 glycoproteins. All T-cells will be positive for this receptor, so this signal will also function as a count of the entire T lymphocyte population within the sample. As such a pivotal event, the signal is from the iFluor® 488 fluorescent probe, arguably the best green fluorescent label currently available, conjugated to an anti-human CD3 antibody (clone SK7), a small 20kD chain. An example of flow cytometry analysis of whole blood cells using this conjugate alone is shown in Figure 2.

The fourth plot is marked by the presence or absence of signal from the labeled anti-CD8 antibody (clone SK1) which is conjugated to APC-iFluor® 750. CD3 is present on all T cells, but CD8 is on T cytotoxic cells specifically, although this moderate-sized 65 kD protein is also expressed in lesser amounts on some other cell types.

CD4 marks the fifth of the possible events, and is present on T helper cells among many others. This 55 kDa transmembrane protein is part of the IgG superfamily, and is also the primary receptor for HIV, playing a role in immunosurveillance for many cell and tissue types. It is detected by an anti-CD4 antibody (clone SK3) tandem-labeled with PE-iFluor® 750.

These PE tandems have the longest emission wavelength present in the kit, with a recommended channel of B13 (772-795 nm). The NIR emission of these tandem dyes, with the strength boosted by either the PE or APC phycobiliproteins, give an easily-identifiable signal. This will provide absolute counts for the T cytotoxic or helper cells present in the sample. When calculating overall results post-gating, these dyes will permit precise demarcation of the two subgroups within the T-cell population. The B-cell population is identified in the sixth possible plot, to detect CD19. This 95kD glycoprotein is expressed on both developing and mature B cells of all types except plasma cells, and will be identified via an anti-CD19 antibody (clone SJ25C1) labeled with APC. This classic red fluorescent label is a common channel preset on almost all flow cytometers.

The final event is to identify NK-cells, and is unique in that it requires the combination of two surface cell antigens, both CD16 and CD56. The NK-cells express both CD56 and CD16 at different levels in their life cycle, thus the kit was designed to detect both. In peripheral blood, the predominant population will express bright CD16, with a dim signal from CD56. For basic whole blood samples, this will be the standard result, whereas NK-cells from secondary lymphoid tissue and from other tissues will primarily show bright CD56. The anti-CD16 antibody is the 3G8 clone, and the anti-CD56 antibody is the MY31 clone. Both antibodies are conjugated with PE, another classic phycobiliprotein. Many instruments have either an automatic PE channel, or can simply be detected with the red laser.

Once the sample has been processed fully, the next phase will be visualizing the results and calculating the absolute cell type concentrations.

Calculations and Example Results

The absolute count for each cell type can be calculated using the following simple equation:

Absolute cell concentration (cells/µL) = [Gated cell count X Total number of beads in tube]/[Number of gated beads X Sample volume aliquoted into tube]

In Figure 1, the simple hierarchy of flow cytometry sorting was outlined. A complete version of gated cell populations as sorted by the flow cytometer is in the table below.

Table. 1 Cell Subsets.

Being able to correctly differentiate not only T, B, and NK cells in general, but also T cell subtypes (i.e. T helper and cytotoxic cells), is useful not only for medical research, but immunological studies in general.

For example, CD3+ CD4+ totals as well as total T-cell and B-cell lymphocyte counts are used as metrics to track the progression of multiple autoimmune diseases. NK-cell quantitation (as counted by CD3- CD16+ and CD56+ populations) is a marker for immune response to tumor formation and growth.

Figure 3. The visualization of a human peripheral blood sample analysis. Human peripheral blood was stained with the ReadiUse™ Human TBNK 6 Color Antibody Kit *Dry Reagent*. Cells were stained at room temperature for 20 minutes, lysed with 1X Lysis/Fix buffer for 15 minutes, and then analyzed by flow cytometry. Live cells were gated on NucPO-1 viability stain. Live cells are gated on CD45+ (2D1)-PerCP-iFluor® 680 for lymphocytes and then gated on CD3+ or CD3- (SK7) iFluor® 488 cell populations. CD3+ cells are shown using CD4 (SK3) PE-iFluor® 750 and CD8 (SK1) APC-iFluor® 750 markers. CD3- cells are shown using CD19 (SJ25C1)-APC and CD56 (5.1H11) / CD16 (B73.1)-PE markers.

Figure 3 shows several dot plots as generated via flow cytometry. Labeled in red, each view is as follows:

• View 1: Plot forward scatter versus side scatter to separate the cells and beads from the bulk of the debris.
• View 2: Plot Anti-CD45 PerCP-iFluor® 680 (675-715 nm) versus side scatter (SSC–Linear scale) for the cell population to separate cells from remaining debris.
• View 3: Plot Anti-CD3 iFluor® 488 fluorescence (515-545 nm) versus side scatter (SSC–Linear scale) for the total cell population to identify the CD3+ T-cell and CD3- lymphocyte populations.
• View 4: Plot Anti-CD8 APC-iFluor® 750 (750-810 nm; 635 nm excitation) versus Anti-CD4 PE-iFluor® 750 (750-810 nm; 488 nm excitation) for the events gated as CD3+ to identify the CD4+ T-cell and CD8+ T-cell populations.
• View 5: Plot Anti-CD19-APC (655-685 nm) versus Anti-CD16/56-PE (562-587 nm) for the CD3- lymphocyte population to identify the B-cell and NK-cell populations.
• Center Top (Viability): Plot NucPO-1 (425-475 nm) versus side scatter for any cell population to determine the viable cells in that population.

For any confusion on the cell groupings as they relate to the sample processing, reference the linear hierarchy (Figure 1) infographic above. Changes to the protocol will of course require commensurate changes to the calculation and examination of assay results.

Conclusion

The ReadiUse™ 6-Color Human TBNK Antibody Kit, with its simplified workflow and lyophilized ingredients for RT storage instead of refrigeration, represents an easier method for lymphocyte immunophenotyping of whole blood or PBMC samples. Accurate quantification of these cell populations is continually necessary in research settings, and by improving the ease of logistical use, this RUO kit provides dependable results with significant time and financial savings.

References

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2. Murray and Nadel's Textbook of Respiratory Medicine (Sixth Edition) Volume 2, 2016, Pages 1624-1638.e4 doi: https://doi.org/10.1016/B978-1-4557-3383-5.00092-0
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4. Yeap, W., Wong, K., Shimasaki, N. et al. CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes. Sci Rep 6, 34310 (2016). https://doi.org/10.1038/srep34310
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